Article Figures & Data
Figures
- Figure 1.
Isoflurane anesthesia does not activate sleep-promoting dFB neurons. A, Tethered flies walking on an air-supported ball were exposed to 2% isoflurane. B, Example movement trace from a single fly following exposure to isoflurane. Loss of movement (orange line) was used as the behavioral endpoint of anesthesia. C, Flies expressing GCaMP6s and CsChrimson in dFB neurons (R23E10-Gal4) were imaged using a 2-photon microscope, with ROIs selected for cell bodies, dendrites, and axons (dFB). Scale bar, 50 µm. D, Mean fluorescence change (dF/F) during optogenetic activation (red bar, top) and corresponding fly movement (bottom). n = 6 flies, 3 stimulations each. E, Mean fluorescence activity before (black) and following loss of movement (orange), normalized to peak optogenetic activation (D). n = 6 flies, two-way ANOVA with Sidak's multiple comparisons. F, Flies expressing GCaMP6s and CsChrimson in EB R3m neurons (R28D01-Gal4), with ROIs selected for cell dendrites, axons (EB), and cell bodies. Scale bar, 50 µm. G, Mean fluorescence change (dF/F) during optogenetic activation (red bar, top) and corresponding fly movement (bottom). n = 9 flies, 3 stimulations each. H, Mean fluorescence activity before (black) and following loss of movement (orange), normalized to peak optogenetic activation (G). n = 9 flies, two-way repeated-measures ANOVA with Sidak's multiple comparisons. Fly image in A from SciDraw. Fly head in C and F created with BioRender.com.
- Figure 2.
The effect of isoflurane on whole-brain neural activity and responsiveness. A, Broad-scale volumetric imaging using a nuclear localized GCaMP was performed during 10 min of spontaneous activity, followed by four 2 s UV visual stimuli and four 0.5 s air puff stimuli. B, Example activity heatmap from 200 randomly selected neurons in a single fly. C, Example neuron from B, showing activity over the recording. Green bars represent periods of detected activity (see Materials and Methods). D, Corresponding behavioral activity for the same fly during the spontaneous period. E, Activity heatmap of the same neurons as in B, under isoflurane anesthesia. F, Activity of the same neuron shown in C, under anesthesia. G, The effect of isoflurane (orange) on activity level (number of frames spent active, left), activity number (% cells active, middle), and behavioral responsiveness as measure by responses to an air puff stimulus (right). n = 14 flies. **p < 0.01 (paired t tests). H, Activity heatmap of responses to the UV stimuli for the same fly shown in B-F. I, Average response profile (purple) for all 345 responding neurons across all 4 stimuli for the fly shown in H. Gray represents individual responses. J, Corresponding behavioral activity for the same fly during the stimulation period. In this example, the fly has not moved. K, Activity heatmap of responses to the UV for the same neurons in H, under isoflurane anesthesia. L, Average response profile for the same neurons, under isoflurane. M, Comparison of mean activity levels (left) and the number of UV-responsive neurons (right) during baseline (black) and isoflurane (orange). n = 13 flies. **p < 0.01; paired t test (activity levels) and two-way repeated-measures ANOVA with Sidak's multiple comparisons (neurons responding) (see Materials and Methods). N, Activity heatmap of responses to the air puff stimuli for the same fly shown in spontaneous and UV panels. O, Example average response profile (blue) for all 34 responding neurons across all 4 stimuli. Gray represents individual responses. P, Corresponding behavior during the 4 air puffs. Q, Activity heatmap of responses to the air puff for the same neurons in N, under isoflurane. R, Average response profile (blue) for the same neurons, under isoflurane. Gray represents individual responses. S, Comparison of mean activity levels (left) and the number of air puff-responsive neurons (right) during baseline (black) and isoflurane (orange). n = 14 flies. *p < 0.05, paired t test (activity levels) and two-way repeated-measures ANOVA with Sidak's multiple comparisons (neurons responding) (see Materials and Methods). Color intensity on heatmaps represents z score fluorescence values, where the darkest are values ≥3, and negative values have been zeroed (white) for display.
- Figure 3.
Neural identity changes under isoflurane anesthesia. A, All UV-responsive cell locations for an example fly during baseline condition. Each dot represents the centroid of the responsive neuron ROI, with the z dimension collapsed. Grayscale background image is a time and z projection of the recording, with minimum and maximum gray values adjusted for clarity. Scale bar, 50 µm. B, All UV-responsive cell locations for the same fly under isoflurane. C, Baseline and isoflurane UV cell locations merged. Green dots represent cells responsive under both conditions. D, Average overlap between baseline and isoflurane conditions for UV-responsive cells for all flies, n = 13. E, All air puff-responsive cell locations for the same fly as A, during baseline condition. F, All air puff-responsive cell locations for the same fly under isoflurane. G, Baseline and isoflurane air puff cell locations merged. In this example, there are no cells in common. H, Average overlap between baseline and isoflurane conditions for air puff-responsive cells for all flies, n = 14. I, Active cell locations for baseline spontaneous condition for the same fly, in a single z slice. J, Active cell locations for isoflurane spontaneous condition, in the same slice. K, Baseline and isoflurane active cells merged. Green dots represent cells active during both conditions. L, Average overlap between baseline and isoflurane conditions for active cells (all slices) for all flies, n = 14.
- Figure 4.
Neural identity changes through time. A, Active cell identity was compared between all pairwise 1 min periods within the spontaneous recording. B, Example active neurons (black dots) for one fly in a single z slice in 1 min bins. Green represents cells active between two bins. Scale bar, 50 µm. C, Pairwise comparison of cell identity overlap between minute bins during baseline and isoflurane anesthesia. n = 14 flies. D, Mean overlap of bins which share the same distance in time apart (1-9 min) for baseline (black) and isoflurane (orange). n = 14 flies. Shaded region represents SEM. E, Self-similarity as an average measure of pairwise overlap for baseline (black) and isoflurane (orange) spontaneous recording. n = 14 flies. **p < 0.01 (paired t test). F, Pairwise comparison of cell identity overlap between minute bins during baseline and induced sleep. n = 8 flies. G, Mean overlap of bins which share the same distance in time (1-6 min) apart for baseline (black) and sleep (red). n = 8 flies. Shaded region represents SEM. H, Self-similarity as an average measure of pairwise overlap for baseline (black) and sleep (red) spontaneous recording. n = 8 flies, paired t test. ns, not significant.
- Figure 5.
Decreased neural connectivity under isoflurane anesthesia. A, Neurons were pairwise correlated to find connected partners. The average number of partners per neuron is the mean degree. B, Example neuron (black) with its two connected partners (top) and their corresponding activity traces (middle). Bottom, Two minutes of correlated activity between two of the neurons. Scale bar, 50 µm. C, Pairwise correlations were performed following the shifting of each neuron trace in time by 1-10 frames, which resulted in changes in connectivity. D, Mean degree changes from baseline (black) to isoflurane (orange). n = 14 flies. **p < 0.01 (paired t test). E, Effect of time shifts on mean degree during baseline (black) and isoflurane (orange). Left, The effect of each shift (frames). Right, Comparison of mean degree without shifts to a 10 frame shift. n = 14 flies. ****p < 0.0001 (two-way repeated-measures ANOVA with Sidak's multiple comparisons between each frameshift to 0). Shaded region represents SEM. F, Mean degree during baseline (black) and induced sleep (red). n = 8 flies, paired t test. G, Effect of time shifts on mean degree during baseline (black) and sleep (red). Left, The effect of each shift (frames). Right, Comparison of mean degree without shifts to a 10 frame shift. n = 8 flies. ****p < 0.0001 (two-way repeated-measures ANOVA with Sidak's multiple comparisons between each frameshift to 0).
- Figure 6.
Isoflurane anesthesia targets transient neural ensembles across the fly brain. A, Example neurons with their significantly correlated partners which can be close (left) or distant (right). Mean distance is calculated from the distances of all partners. Scale bar, 50 µm. B, Normalized degree and average corresponding distance to correlated partners for each significant neuron in an example fly (right) during baseline (black) and isoflurane (orange). This example is a case where neurons with higher degree tend to be closer together (left) under isoflurane. C, Normalized degree and distance of each neuron in a different fly (right), where the example shows neurons with higher degrees tend to be further apart under isoflurane (left). D, Normalized degree and distance of each neuron in an example fly where distance between connected neurons does not generally change under isoflurane; however, degree decreases. E, The effect of neuron distance (close, medium, and far; see B, right) on connectivity (degree) during baseline wakefulness (black) and during anesthesia (orange). n = 14 flies. **p < 0.01 (two-way repeated-measures ANOVA with Sidak's multiple comparisons). F, The effect of degree (low and high; see B, right) on neuron distance. n = 14 flies, two-way repeated-measures ANOVA with Sidak's multiple comparisons. G, Connectivity during baseline (black) and anesthesia (orange) in neurons active across both conditions (overlap), and neurons active only within a condition (nonoverlap). n = 14 flies. **p < 0.01 (two-way repeated-measures ANOVA with Sidak's multiple comparisons). H, The effect of neuron distance (close, medium, and far) on connectivity (degree) during baseline wakefulness (black) and during sleep (red). n = 8 flies, mixed-effects model with Sidak's multiple comparisons. I, The effect of degree (low and high) on neuron distance. n = 8 flies. *p < 0.05 (two-way repeated-measures ANOVA with Sidak's multiple comparisons). J, Connectivity during baseline (black) and sleep (red) in neurons active across both conditions (overlap), and neurons active only within a condition (nonoverlap). n = 8 flies, two-way repeated-measures ANOVA with Sidak's multiple comparisons.
Tables
Reagent Citation Expression/use 23E10-Gal4 Jenett et al., 2012 dFB driver 28D01-Gal4 Jenett et al., 2012 R3m EB ring neuron driver 57C10-Gal4 Jenett et al., 2012 Pan-neuronal driver 57C10-LexA Jenett et al., 2012 Pan-neuronal driver UAS-CsChrimson Klapoetke et al., 2014 Optogenetic activation UAS-Chrimson88 Strother et al., 2017 Optogenetic activation UAS-GCaMP6s Chen et al., 2013 Fluorescent calcium indicator, cytosolic expression LexAop-nlsGCaMP6f Tainton-Heap et al., 2021; provided by Barry J Dickson Fluorescent calcium indicator, nuclear localized expression
